Summary
Stable transformation of maize, Chlamydomonas reinhardtii, and Saccharomyces
cerevisiae nuclear genomes, as well as C. reinhardtii chloroplasts and
S. cerevisiae mitochondria was achieved by particle bombardment with the
PDS-1000/He instrument using plasmid DNA coated onto gold or tungsten
microparticles. Results demonstrate that the kind and size of microparticles
are important factors in determining the efficiency of transformation.
Maize callus is transformed approximately five-fold more efficiently with
0.6 gold particles than with 1 gold particles. Nuclear transformation
of S. cerevisiae is over ten-fold more efficient using 0.6 gold particles
than with 1 gold particles. Yeast mitochondrial transformants, which
arise at a very low frequency using 0.6 gold particles, were not found
using larger gold particles or M5 tungsten particles. Nuclear transformation
of Chlamydomonas was 2.5-fold more efficient with 0.6 gold particles
than with either 1 gold or M10 tu
electron microscopy. Accurate size analysis of gold
microparticles is particularly difficult because of their high density
and their potential to agglomerate. Most suppliers of gold powder determine
particle size by sedimentation techniques. This procedure has the advantage
of analyzing a very large number of particles as well as being a rapid
and relatively simple procedure. However, sedimentation analysis does
not distinguish between individual and agglomerated particles, and particle
diameter is estimated assuming perfectly spherical particles. While electron
microscopy is more labor intensive, it permits visual determination of
the particle shape and allows direct measurement of the diameter of individual
particles. Therefore, non-spherical batches of gold particles can be excluded
from further assay and agglomerated particles can be eliminated from the
size calculation for maximum consistency between samples. Figure 1 shows
scanning electron micrographs of gold and tungsten particles representative
of the those sold by Bio-Rad. Also shown are size distributions for the
four sets of particles used in these experiments. The contrast between
the two types of particles in terms of the size and shape is particularly
evident. Scanning electron microscopy has been used as a part of the quality
control procedure to determine the average size, the variation in size,
and the sphericity of Bio-Rad gold particles since 1993. Information on
the average particle diameter, the standard deviation of the diameter,
and the aspect ratio (the ratio of the long axis of the particle to the
short axis of the particle a measure of sphericity) is provided with
every lot of gold microparticles. Additionally, quality control includes
a biological assay using the particles in bombardment and measuring transient
or stable expression. Together, these assays are designed to assure lot-to-lot
consistency of gold particles for Biolistic applications.

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ngsten particles. Using 0.6 gold
particles, the transformation efficiency of Chlamydomonas chloroplasts
was 2.5-fold higher than with M10 tungsten particles and 4-fold higher
than with 1 gold particles.

Introduction
Biolistic technology, or particle bombardment, is a physical method of
introducing DNA into cells. In theory, all cells should be transformable
by this method. The technique was originally developed to transform monocotyledonous
plants, and has since been used to transform numerous species of both
mono- and dicotyledenous plants (Klein, et al., 1990). Particle bombardment
has also been used to transform a wide variety of tissue culture cells
and animal organs (Johnston & Tang, 1993). Additionally, the technique
has been used to transform bacteria and subcellular organelles (Boynton
& Gillham, 1993, 1996; Butow & Fox, 1990; Smith, et al., 1992).

Transformation of all these different cell types requires optimization
of the physical parameters used in bombardment (Sanford, et al., 1993).
The parameters which have the greatest effect on transformation efficiency
include the vacuum in the bombardment chamber, the distance the particles
travel before striking the target cells, and the size and density of the
particles used in bombardment. Of less importance are the helium pressure,
the gap distance (the distance between the rupture disk and the macrocarrier),
and the macrocarrier travel distance. For most applications, a helium
pressure of about 1,100 psi, a gap distance of 510 mm, and a macrocarrier
travel distance of about 8 mm are near optimal. Each of these parameters
affects the particle velocity and each interacts with the others. The
greater the particle
velocity, the more likely the particle will penetrate
the cell; this is particularly important when using small particles. The
chamber vacuum affects the velocity of the microparticles by reducing
the drag on the particles as they travel toward the target tissue: the
higher the chamber vacuum, the less the particles will be decelerated.
The distance between the stopping screen and the target tissue has several
effects on the transformation efficiency. The longer the travel distance,
the greater the spread of the particles over the target and the less pronounced
is the helium shock wave striking the cells. On the other hand, a longer
travel distance results in reduced particle speed and a decreased likelihood
that a particle will penetrate the target.

Because the likelihood of a subcellular-sized particle penetrating a
cell is proportional to the kinetic energy of the particle, materials
of high density have generally been preferred for use in biolistic technology.
These compounds include tungsten, platinum, and gold. Of these, gold is
favored because it is biologically inert and because spherical gold particles
can be produced in a narrow size range. Physical parameters of gold particles
are presented in Table 1. Note the nearly five-fold increase in the number
of particles per gram for the 0.6 gold relative to the 1.0 gold. The
importance of particle size and density on transient expression has been
demonstrated for a variety of cell systems (Sanford, et al., 1993). Here
we present data to demonstrate that for stable expression in several biological
systems, particle bombardment is more efficient using 0.6 gold particles
than using particles of larger size.

Methods and R
esultsStable transformation of Type I callus of Zea mays. Maize Type I callus
(Wan, et al., 1995) was subjected to osmotic pretreatment by placing the
tissue on high sucrose media for 4 hr. prior to bombardment. Tissues were
bombarded in the PDS- 1000/He with 1 or 0.6 gold particles coated
with pBC17 (Goff, et al., 1990), a plasmid which encodes genes for the
C1 regulatory gene and the B-Peru allele of the B regulatory gene in the
maize anthocyanin biosynthetic pathway. When expressed together, these
genes activate this pathway and give rise to pigmented cells. Bombardment
conditions were as follows. Each plate was shot twice. Each shot contained
0.06 mg or 0.5 mg of 0.6 gold particles, or 0.5 mg of 1 gold particles,
and all particles were coated with 0.33 g of plasmid pBC17; 16 target
tissue pieces were placed in a ring on a 100 mm plate; 3 plates were shot
per treatment; a stainless steel 200 x 200 wire mesh screen (McMaster-Carr,
cat. no. 9236T11) was inserted at Level 1 as a post-launch baffle (Russell,
et al., 1992) to reduce cell damage and the tissue sections were placed
at Target Level 2; the bombardment chamber was reduced to 28 Hg vacuum
and cells were bombarded at a helium pressure of 650 or 900 psi.

Transient expression and target tissue damage were qualitatively assessed
4 days after gene delivery. Generally, bombardment with 1 mg of 1 gold
particles produced relatively high levels of transient expression as judged
by observation of pigmented cells. The level of transient expression in
cells bombarded with 0.6 gold particles was dependent on the amount
of gold used in bombardment. Reducing the amount of gold eight-fold, from
1 mg to 0.12 mg, resulted in an eightfold decre
ase in the number of gold
particles and in a proportional decrease in the number of transiently
expressing cells; plates bombarded with 1 mg of 0.6 gold particles had
more transiently-expressing anthocyanin cells than did plates bombarded
with 1 mg of 1 particles. Tissue damage was less in cells bombarded
with 0.6 gold than in tissue bombarded with the same amount of 1 gold.
Although the presence of the wire mesh did not affect the number of cells
that transiently express anthocyanin, it did reduce tissue damage and
resulted in higher numbers of stable events. Results of stable transformation
are presented in Table 2. Transformation was assessed by counting the
number of individual multi-celled red-pigmented sectors present after
6 weeks of growth on non-selective media. Stable sectors are derived from
cells which have been transformed, divide, and continue to express the
anthocyanin gene in the daughter cells. Each stable sector has the potential
of growing into a mature plant expressing the transforming gene. Compared
to callus tissue bombarded with 1 gold, four- to eight-fold more red
sectors were present in tissue bombarded with 0.6 gold, although in
one experiment eight-fold less gold (and hence, eight-fold fewer gold
particles) was used than in the other experiment (see Table 2). These
results indicate that the level of transient expression is not always
an accurate indication of the level of stable expression. Since the experiments
summarized in Table 2 were carried out at different times and with different
callus culture lines, the absolute numbers of transformants should not
be compared between Experiment 1 and Experiment 2.

For nuclear transformation, arg+ colonies appeared within 7 days post-bombardment,
while photosynthetic colonies resulting from chloroplast transformation
were visible 35 days postbombardment. In these experiments, final scoring
of all transformants was performed
1314 days after bombardment. Results
of several bombardments are summarized in Table 3. Both nuclear and chloroplast
transformation efficiencies were on average 2.5-fold higher using 0.6
gold particles compared to using M10 tungsten particles. In a single
experiment, nuclear transformation of Chlamydomonas was about two-fold
lower using 1 gold particles than using 0.6 gold particles, while
the level of Chlamydomonas chloroplast transformation was four-fold lower
using 1 gold particles than using 0.6 gold particles.

Stable transformation of yeast mitochondria. A derivative of Saccharomyces
cerevisiae strain MCC109 (MATα ade2-101 ura3-52 kar1-1)
lacking mitochondrial DNA [rhoo] was used for assaying nuclear and mitochondrial
transformation. Cells were bombarded with particles carrying the URA3
gene on plasmid YEp352 and a plasmid carrying intron 2 from the COXI gene
(either pJVM161 or pJVM164; Moran, et al., 1995) and Ura+ transformants
were selected on media lacking uracil. Mitochondrial transformants were
identified by mating the [rhoo] Ura+ nuclear transformants with a respiration-deficient
mutant (S. cerevisiae strain 161) containing a deletion within
intron 2 of the COXI gene (Zimmerly, et al., 1995) and screening for progeny
capable of respiring on glycerol-containing media. Recombination between
the defective portion of the COXI gene present in S. cerevisiae
strain 161 and the COXI gene from the plasmid present in the mitochondria
of the transformant restores respiratory growth. Bombardment conditions
were as follows: 0.6 mg of gold or M5 tungsten (average size = 0.4 )
were coated with a mixture of 8 g of COXI mitochondrial plasmid D
NA and
1.6 g of YEp352 plasmid; 2 x 108 cells were spread on 100 mm agar plates;
5 plates were shot for each experimental condition; the bombardment chamber
was reduced to 28 Hg vacuum and cells were bombarded at Target Level
2 at a helium pressure of 1,1001,300 psi.

Nuclear transformation was quantitated by counting Ura+ transformants
5 days post-bombardment following growth of transformed cells on uracil-deficient
media. Ura+ transformants were then analyzed for mitochondrial transformation
by mating to S. cerevisiae strain 161 on glucose-containing media (Anziano
& Butow, 1991; Butow, et al., 1996). Mitochondrial transformants were
identified by replica plating these colonies onto media containing glycerol.
Results are summarized in Table 4. Nuclear transformation is quite efficient
using either 0.6 gold particles or M5 tungsten to deliver the DNA; there
is a greater than 10- fold decrease in the number of transformants when
1 gold particles are substituted for 0.6 gold particles. In these
experiments, mitochondrial transformants were only isolated when sub-micron
sized gold particles were used. While mitochondrial transformants have
been isolated using larger tungsten particles, results with those particles
were extremely erratic in our hands, even in terms of nuclear transformation.
Similar results were found when bombardment was performed with the Ura+
and COXI sequences on a single plasmid (results not shown).

Conclusions
Results presented here show that particle size is an important factor
for optimizing Biolistic transformation. Maximum stable transformation
of cellular organelles (chloroplasts and mitochondria
) and certain cell
types (Chlamydomonas, yeast, and maize callus) occurs using sub-micron
sized gold particles. Smaller particles are probably more efficient than
larger particles for Biolistic transformation in these systems because
they cause less cell damage, resulting in better survival. Organelles
could be irreversibly destroyed upon impact with large particles. In the
case of maize callus, while cells bombarded with both 0.6 and 1.0
gold particles transiently express equivalent levels of anthocyanin four
days postbombardment, cells bombarded with the smaller particles gave
rise to many more stable transformants, most likely because they sustained
less cell damage which could be more easily repaired.

Advantages of gold microparticles include their uniform size and shape
and their biological inertness. In contrast, tungsten microparticles are
irregular in shape and heterogeneous in size, can be toxic to cells, are
subject to surface oxidation that can alter DNA binding, and, over time,
can catalytically degrade DNA bound to them (Sanford, et al., 1993). Thus,
because of higher transformation efficiency of gold particles, greater
variability in results using tungsten, and the need for special precautions
in particle preparation, handling, and transformation procedures when
using tungsten, gold particles are superior to tungsten particles for
Biolistic applications. 0.6 gold microparticles can be used with the
Yeast Optimization Kit (catalog number 170-3100) to introduce the novice
to the principles of particle bombardment.

The size of small particles may be determined by several methods, including
molecular sieving, sedimentation, light scattering, electrical sensing,
optical sensing, and'"/>

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